Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling.
The aim of this study was to determine whether primary human tubular cell monolayers could provide a powerful tool with which to investigate the renal proximal tubular handling of xenobiotics. Human proximal and distal tubule/collecting duct cells were grown as monolayers on permeable filter supports. After 10 days in culture, proximal tubule cells remained differentiated and expressed a wide palette of transporters at the mRNA level including NaPi-IIa, SGLT1, SGLT2, OCT2, OCTN2, OAT1, OAT3, OAT4, MDR1, MRP2 and BCRP. At the protein level, the expression of a subset of transporters including NaPi-IIa, OAT1 and OAT3 was demonstrated using immunohistochemistry. Analysis of the expression of the ATP binding cassette efflux pumps MDR1, MRP2 and BCRP confirmed their apical membrane localisation. At the functional level, tubule cell monolayers retain the necessary machinery to mediate the net secretion of the prototypic substrates; PAH and creatinine. PAH secretion across the monolayer consisted of the uptake of PAH across the basolateral membrane by OAT1 and OAT3 and the apical exit of PAH by a probenecid and MK571-sensitive route consistent with actions of MRP2 or MRP4. Creatinine secretion was by OCT2-mediated uptake at the basolateral membrane and via MDR1 at the apical membrane. Functional expression of MDR1 and BCRP at the apical membrane was also demonstrated using a Hoechst 33342 dye. Similarly, measurement of calcein efflux demonstrated the functional expression of MRP2 at the apical membrane of cell monolayers. In conclusion, human tubular cell monolayers provide a powerful tool to investigate renal xenobiotic handling.
Brown
Colin D A
CD
Institute for Cell & Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne, UK. c.d.a.brown@ncl.ac.uk
Sayer
Rachel
R
Windass
Amy S
AS
Haslam
Iain S
IS
De Broe
Marc E
ME
D'Haese
Patrick C
PC
Verhulst
Anja
A
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003404:Creatinine / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008954:Models, Biological; D027361:Organic Anion Transporters / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015262:Xenobiotics / Q000493:pharmacokinetics / Q000633:toxicity*
D018528:ATP-Binding Cassette Transporters; D027361:Organic Anion Transporters; D015262:Xenobiotics; D003404:Creatinine